JP6259245B2 - Peptide having affinity for immunoglobulin G and IgG type antibody adsorbent using the same - Google Patents

Description

  The present invention relates to a peptide having affinity for immunoglobulin G and an IgG antibody adsorbent using the peptide.

  Recent advances in medicine, especially internal medicine, hematology, immunology, and clinical laboratory science, have revealed malignant substances in blood that are thought to be closely related to the cause of the disease or the progression of the disease. It is becoming. For example, autoantibodies and immune complexes based on immunoglobulin G (IgG) present in blood as a main cause of autoimmune diseases related to biological immune functions such as rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis And is thought to be closely related to the progression of the disease state.

  Therefore, by specifically adsorbing and removing the autoantibodies and immune complexes from body fluid components such as blood and plasma, the progression of the above-mentioned diseases can be prevented, the symptoms of the diseases can be reduced, and further the healing of the diseases can be achieved. It is expected to be accelerated. As a technique for removing autoantibodies and immune complexes in body fluids, whole blood is separated into blood cell components and plasma components, and the autoantibodies and immune complexes contained in plasma are adsorbed by an adsorbent and removed. There is a way.

Japanese Patent Publication No. 63-053971 Japanese Patent Publication No. 3-065190 Japanese Patent No. 4394932

Future Materials, Vol.8, No.11 (2008), pp28-34 Bulletin of the American Academy of Sciences, Volume 93 (1996), pp5688-5692

  Conventionally, as an adsorbent used for such a purpose, an adsorbent in which tryptophan, which is a hydrophobic amino acid, is immobilized as a ligand on a water-insoluble carrier is known (Patent Document 1). Furthermore, biologically derived IgG-type antibody binding substances such as protein A and anti-human immunoglobulin antibodies known to specifically interact with IgG-type antibodies, so-called biological ligands, are immobilized on a water-insoluble carrier. An adsorbed material is known (Non-Patent Document 1).

  However, while an adsorbent in which tryptophan is immobilized on a water-insoluble carrier as a ligand has a therapeutic effect, it cannot be said that the capacity for adsorbing IgG-type antibodies is sufficiently large. Furthermore, the adsorbent has the disadvantage that it also removes fibrinogen (Fbg), which is not a pathogenic substance, and has poor selective adsorption.

  On the other hand, an adsorbent in which protein A, which is an example of a biological ligand, is immobilized on a water-insoluble carrier has specific adsorption performance for IgG antibodies (Patent Document 2). Therefore, it is considered that the adsorbent can solve the above-described problems of the adsorbent having tryptophan as a ligand. However, these biological ligands generally have the disadvantage that it is difficult to secure raw materials and the product cost is high. Furthermore, when the adsorbent is used in contact with body fluids, there is a risk that the biological ligand will elute into the body fluids and cause serious side effects. In addition, there is a drawback that there is a risk of infection from unknown viruses or pathogens that may be introduced in the manufacturing process of the biological ligand.

  With the recent development of molecular biology and instrumental analysis technology, detailed structural information of proteins and their functions have been elucidated, and it has become possible to achieve the functions of huge proteins with small peptides. For example, peptides having a helix-loop-helix structure and a specific binding function similar to antibodies have been found. As such a peptide, Patent Document 3 reports a synthetic peptide that binds to a granulocyte colony stimulating factor (G-CSF) receptor. However, no binding between a synthetic peptide having a helix-loop-helix structure and an IgG type antibody has been reported.

  The present invention has been made in view of the above circumstances, a peptide capable of highly selectively adsorbing an IgG-type antibody with a high adsorption capacity, an IgG-type antibody adsorbent using the peptide, and an adsorbent thereof It is an object of the present invention to provide an IgG type antibody adsorber filled with, and a body fluid purification device including the adsorber.

  As a result of intensive studies to solve the above problems, the present inventors have found that a peptide having a specific amino acid sequence having a helix, loop, and helix structure selectively binds to an IgG antibody. Furthermore, the present inventors use a therapeutic device (for body fluid purification) as an adsorbent in which the peptide is used as a ligand for binding an IgG antibody, and the ligand is immobilized on a water-insoluble carrier. As a treatment device, a body fluid purification device), it has been found that IgG antibodies constituting autoantibodies and immune complexes are adsorbed with surprisingly high adsorption capacity and high selectivity from body fluids such as blood, plasma and serum. The present invention has been completed.

That is, the present invention relates to the following.
[1]
A peptide having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
[2]
The peptide according to [1], which has the amino acid sequence set forth in SEQ ID NO: 2 and wherein the seventh cysteine residue and the 45th cysteine residue in the amino acid sequence form a disulfide bond with each other.
[3]
A water-insoluble carrier;
A peptide immobilized on the water-insoluble carrier,
An IgG type antibody adsorbent, wherein the peptide is the peptide according to [1] or [2].
[4]
The IgG-type antibody adsorbent according to [3], wherein the peptide is immobilized on the water-insoluble carrier directly or via a linker by a mercapto group of at least one cysteine residue of the peptide.
[5]
The IgG-type antibody adsorption according to [3], wherein the peptide is immobilized on the water-insoluble carrier directly or via a linker with an amino group in a side chain of at least one lysine residue of the peptide. Wood.
[6]
The peptide according to [3], wherein the peptide is immobilized on the water-insoluble carrier directly or via a linker by a carboxyl group of a side chain of at least one glutamic acid residue or aspartic acid residue of the peptide. IgG type antibody adsorbent.
[7]
The IgG-type antibody adsorbent according to [3], wherein the peptide is immobilized on the water-insoluble carrier directly or via a linker by a carboxyl group at the carboxy terminus of the peptide.
[8]
The IgG-type antibody adsorbent according to [3], wherein the peptide is immobilized on the water-insoluble carrier directly or via a linker by an amino group at the amino terminal of the peptide.
[9]
The IgG-type antibody adsorbent according to any one of [4] to [8], wherein the linker is oligoethylene glycol or polyethylene glycol.
[10]
The IgG-type antibody adsorbent according to any one of [3] to [9], wherein the peptide is immobilized in an amount of 0.1 to 100 μmol per mL of the water-insoluble carrier.
[11]
The IgG-type antibody adsorbent according to any one of [3] to [10], wherein the water-insoluble carrier is a porous body on particles, and the water-insoluble carrier has an exclusion limit molecular weight of 150,000 to 10 million. .
[12]
A container having an inlet port through which a liquid flows and an outlet port through which the liquid flows out;
An IgG type antibody adsorber comprising the IgG type antibody adsorbent according to any one of [3] to [11] filled in the container.
[13]
A body fluid purification device comprising the IgG antibody adsorber according to [12],
A body fluid purification device, wherein the liquid flowing into the IgG antibody adsorber is a body fluid selected from the group consisting of blood, plasma, and serum.

  The peptide of the present invention can adsorb IgG-type antibodies with high adsorption capacity and high selectivity.

  Furthermore, since the three-dimensional structure of the peptide is a helix, loop, and helix structure, it is stable even in body fluids. Therefore, the adsorbent in which the above peptide is immobilized on a water-insoluble carrier can adsorb IgG with high selectivity and high selectivity from body fluids such as plasma and serum.

It is a schematic cross section showing one embodiment of an extracorporeal circulation module using an IgG type antibody adsorbent. It is a graph which shows the adsorption rate of the protein in each support | carrier manufactured in the Example.

  The present invention will be described in detail below. The following embodiment is an example for explaining the present invention, and is not intended to limit the present invention to this embodiment alone.

  The “IgG type antibody” in the present embodiment may be a protein in which a region other than the Fc region in the protein molecule is denatured and / or deleted as long as it is a protein having the same Fc region as the IgG class. .

  In the present specification, various amino acid residues are described in one-letter code according to conventional methods in the field, and the amino acid sequence of the peptide is described from the amino terminus to the carboxy terminus from left to right.

  The peptide of the present invention has the amino acid sequence described in SEQ ID NO: 1 (FNMQQQRRFYEALHEAAAAAGGGGGGGNEEQRNAKIKSIRDDC) or SEQ ID NO: 2 (CGFNMQCQRRFYEALHEAAAAAGGGGGGGNEEEQRNAKIKSIRDDC). The peptide can be used as a ligand.

  The peptide may be any peptide as long as it has the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2 continuously without any deletion, but preferably the peptide described in SEQ ID NO: 1 or SEQ ID NO: 2 A peptide consisting of an amino acid sequence. Further, it preferably has the amino acid sequence set forth in SEQ ID NO: 2, and the seventh cysteine residue and the 45th cysteine residue in the amino acid sequence preferably form a disulfide bond with each other.

  The total length of the peptide is preferably 43 to 200 amino acid residues, and more preferably 43 to 100 amino acid residues.

  Of the above peptides, acyclic peptides can be synthesized according to peptide synthesis methods known per se in the art. Examples thereof include liquid phase synthesis methods such as Fmoc solid phase synthesis method and fragment condensation method. In view of simple operation, the peptide synthesis method is preferably a solid phase synthesis method. In addition, among the peptides of the present invention, a cyclic peptide can be obtained by obtaining a non-cyclic peptide and then cyclizing it.

  Acyclic peptides can be produced not only by chemical synthesis methods such as the above-mentioned solid phase synthesis method and liquid phase synthesis method, but also by genetic engineering techniques. For example, a nucleic acid having a base sequence encoding the amino acid sequence described above is incorporated into an appropriate expression vector, and an appropriate host cell (eg, mammalian cell, insect cell, E. coli) is transformed with the obtained expression vector. Then, the peptide can be obtained from the culture by culturing the host cell under conditions suitable for the expression of the peptide.

  The peptide can be used as an IgG-type antibody adsorbent by fixing it to a water-insoluble carrier.

  A water-insoluble carrier means a carrier that is insoluble in water.

  The carrier used for immobilizing the ligand is a reactive functional group such as an amino group, a carboxyl group, or a hydroxyl group that has a hydrophilic surface and can be used to form a covalent bond with the ligand. Those having a group are preferred. The water-insoluble carrier is preferably a porous material having a large effective surface area that can be used for adsorption (porous material).

  As the water-insoluble carrier, any known shape such as a particle shape, a fiber shape, a sheet shape, and a hollow fiber shape can be used. In consideration of the amount of ligand retained or the handleability as an adsorbent, the water-insoluble carrier is preferably in the form of particles.

  The average particle size of the spherical carrier or the particulate carrier is preferably 25 μm to 2500 μm. From the viewpoint of the specific surface area of the carrier and the fluidity of the body fluid, the average particle size is more preferably 50 μm to 1500 μm.

  As the carrier that can be used, organic or inorganic porous materials such as cellulose gel, dextran gel, agarose gel, polyacrylamide gel, porous glass, vinyl polymer, etc. can be used, and carriers used for ordinary affinity chromatography All materials for can be used.

Examples of these carriers are cellulosic carriers such as Asahi Kasei Microcarrier (manufactured by Asahi Kasei Co., Ltd.) and CM-Cellulofine (registered trademark) CH (exclusion limit protein molecular weight: about 3 × 10 ⁶ , sold by Seikagaku Corporation). JP-A-1-044725, a total porous activated gel, a polyvinyl carrier such as CM-Toyopearl (registered trademark) 650C (exclusion limit protein molecular weight: 5 × 10 ⁶ , manufactured by Tosoh Corporation), CM-Tris Polyacrylamide-based carriers such as acrylic M (CM-Triacryl M) [exclusion limit protein molecular weight: 1 × 10 ⁷ , Pharmacia-LKB, Sweden], and Sepharose CL-4B (Sepharose CL-4B) [exclusion limit protein molecular weight: 2 × 10 ^7, Sul Den Country Pharmacia -LKB (Pharmacia-LKB) Co. Ltd.] organic carriers agarose-based carrier such as, well, CPG-10-1000 [exclusion limit protein molecular weight: 1 × 10 ^8, the average pore diameter: 100 nm, USA Electro chromatography And inorganic carriers such as porous glass such as those manufactured by Electro-Nucleonics.

  The water-insoluble carrier is preferably a body-insoluble water-insoluble carrier. A water-insoluble carrier for body fluid purification is a carrier that does not dissolve in body fluids when the adsorbent comes in contact with body fluids such as blood, has little nonspecific adsorption of proteins, has a low ability to produce bradykinin, and has a complement activation ability Means a carrier having high fluid compatibility such as blood compatibility. The water-insoluble carrier for body fluid purification is preferably a crosslinked polyvinyl alcohol (crosslinked PVA).

  As the water-insoluble carrier, a particulate porous body is preferable, and for example, porous polymer particles can be used. The particulate porous body can immobilize the ligand. Since the molecular weight of the IgG type antibody that is the target adsorbing substance is about 150,000, the exclusion limit molecular weight (protein) of the particulate porous material is preferably 150,000 to 10 million. The most preferred exclusion limit molecular weight is 1 million to 5 million.

  The polymer composition may be a known polymer that can have a porous structure, such as a polyamide polymer, a polyester polymer, a polyurethane polymer, and a vinyl compound polymer. In particular, a vinyl compound-based porous polymer hydrophilized with a hydrophilic monomer gives preferable results.

  As a method for supporting the ligand on the water-insoluble carrier, a known carrier activation method and immobilization method can be used. For example, when the carrier has an amino group or a carboxyl group, a method of performing a condensation reaction with a ligand in the presence of a condensation reagent such as dicyclohexylcarbodiimide, and two or more functional groups such as glutaraldehyde with the carrier and the ligand And a method of bonding using a compound having the above. In addition, when the carrier has a hydroxyl group, for example, a method in which cyanogen halide such as cyanogen bromide acts on the carrier and reacts with the amino group portion of the ligand, and an epoxy group such as epichlorohydrin is included. Examples thereof include a method in which a compound is allowed to act on a carrier and reacted with the amino group or mercapto group of the ligand. When the ligand has cysteine, it can be immobilized by reacting with a maleimidized carrier.

  If necessary, a material in which a molecule (linker) having an arbitrary length is introduced between the water-insoluble carrier and the ligand can be used as the adsorbent. Examples of the linker molecule include a polymethylene chain, an oligoethylene glycol chain, a polyethylene glycol chain, and a polyalanine chain. For example, the hydroxyl group of agarose and the glycidyl group on one side of polyethylene glycol diglycidyl ether can be reacted and bonded, and the remaining glycidyl group and the ligand can be reacted and bonded.

  In this embodiment, the peptide may be immobilized on a water-insoluble carrier directly or via a linker by a mercapto group of at least one cysteine residue that the peptide has, or the peptide has at least one peptide that the peptide has. It may be immobilized on a water-insoluble carrier directly or through a linker by the amino group of the side chain of one lysine residue, or the peptide has at least one glutamic acid residue or aspartic acid residue of the peptide. It may be immobilized on a water-insoluble carrier directly or via a linker by a side chain carboxyl group. In addition, the peptide may be immobilized on the water-insoluble carrier directly or via a linker by a carboxyl group at the carboxy terminus of the peptide, or the peptide may be directly or via a linker by the amino group at the amino terminus of the peptide. And may be immobilized on a water-insoluble carrier.

  If the amount of the peptide immobilized on the water-insoluble carrier is too small, the adsorption ability tends to be low, and if it is too large, the increase in the adsorption capacity is saturated. Therefore, the range of 0.1 to 100 μmol per 1 mL of water-insoluble carrier is preferable, and the range of 1 to 50 μmol is more preferable. The amount of the peptide immobilized is calculated based on the difference in absorbance before and after the reaction by measuring the absorbance of the reaction solution before and after the peptide introduction reaction with an ultraviolet-visible spectrophotometer.

  The IgG-type antibody adsorbent can be used as an IgG-type antibody adsorber by being filled and held in a container having an inlet port through which a liquid flows and an outlet port through which the liquid flows out. Examples of such an IgG type antibody adsorber include an extracorporeal circulation module using an IgG type antibody adsorbent.

  FIG. 1 is a schematic cross-sectional view showing an embodiment of an extracorporeal circulation module using an IgG type antibody adsorbent. In the extracorporeal circulation module 1 shown in FIG. 1, a cap 6 having an inlet port 5 (introduction port) through which a body fluid flows is inserted into one end opening of a cylinder 2 through a packing 4 with a filter 3 on the inside. Then, a cap 8 having an outlet port 7 (outlet port) through which a body fluid flows out through a packing 4 ′ with a filter 3 ′ on the inside of the other end opening of the cylinder 2 is screwed to form a container, and a filter is formed. The adsorbent layer 9 is formed by filling and holding the IgG type antibody adsorbent in the gap between 3 and 3 ′. The extracorporeal circulation module 1 can be used, for example, as a body fluid purification device for removing autoantibodies and immune complexes.

  The adsorbent layer 9 may be filled with an IgG type antibody adsorbent alone, or may be mixed with other adsorbents and filled. Alternatively, the IgG type antibody adsorbent and other adsorbent may be stacked so as to be laminated. As another adsorbent, for example, activated carbon having a wide range of adsorbability can be used. This can be expected to have a wide range of clinical effects due to the synergistic effect of the adsorbent. The volume of the adsorbent layer 9 is appropriately about 50 mL to 700 mL when used for extracorporeal circulation.

  When using a body fluid purification device for extracorporeal circulation, there are two main methods. One method is to separate the blood taken out from the body into a plasma component and a blood cell component using a centrifuge or a membrane plasma separator, and then pass the plasma component through the body fluid purification device and purify it. It is a method of returning to the body together with blood cell components. Another method is a method in which blood taken out from the body is directly passed through the body fluid purification device for purification.

  As a method for passing body fluid, it may be continuously passed or used intermittently depending on clinical necessity or according to the installation situation of equipment.

  Such an IgG type antibody adsorbent of the present invention can also be grasped as an IgG type antibody adsorbent for body fluid purification devices. In addition, you may use the IgG type antibody adsorption material and IgG type antibody adsorption device of this invention as an IgG type autoantibody adsorption material and an IgG type autoantibody adsorption device. As used herein, “autoantibody” means an antibody produced against autologous cells or tissues (including transplanted ones).

  IgG type antibody adsorbent is not only used as a treatment device by filling in an apparatus, but also an adsorbent for separation such as immunoglobulin, autoantibody, immunoglobulin derivative, immunoglobulin complex, adsorbent for purification, and these It can also be used very effectively as a measurement substrate.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these.

Example 1
1. Peptide synthesis method N, N-dimethylformamide (DMF) was added to 3 g (0.48 mmol) of Fmoc-PAL-PEG-PS resin (Life Technology, substitution rate 0.16 mol / g) and sufficiently swollen. . After adding 20 mL of 30% piperidine to the suspension containing the swollen resin and shaking for 1 minute, 20 mL of 30% piperidine was added again and shaken for 10 minutes. After thoroughly washing the resin with DMF, 2 μL each of 1% 2,4,6-trinitrobenzenesulfonic acid (TNBS) and 10% N, N-diisopropylethylamine (DIEA) was added to the resin taken out. It was judged that the Fmoc group was deprotected and the amino group was exposed by confirming that the resin was colored in red after standing for 5 minutes. Fmoc-amino acid 4.8 mmol, O- (6-chlorobenzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HCTU) 4.8 mmol, DIEA 9.6 mmol Amino acid was added by adding 20 mL of the mixture to the resin and shaking for 30 minutes at room temperature. After washing the resin with DMF, 2 μL each of 1% TNBS and 10% DIEA was added to a small amount of resin and allowed to stand for 5 minutes to confirm that the resin did not turn red, and all amino groups were reacted. It was judged. 20 mL of 5% acetic anhydride was added to the resin and shaken for 5 minutes to acetylate unreacted amino groups. By repeating this process, the target peptide was synthesized.

  The peptide-synthesized resin was washed with diethyl ether and dried. 40 mL of cleaved solution adjusted to trifluoroacetic acid (TFA): anisole: ethyl methyl sulfide: 1.2-ethanedithiol = 93: 3: 3: 1 (volume ratio) was added and left at room temperature for 4 hours for deprotection. And de-resin reaction. Ice-cooled diethyl ether was added to the resulting supernatant to precipitate the peptide, which was centrifuged and the supernatant was discarded. The obtained precipitate was washed several times with diethyl ether and then sufficiently dried. The obtained peptide was dissolved in 0.1% TFA aqueous solution, and the target peptide was purified by reverse phase HPLC.

  Next, the purified peptide was stirred dropwise over 100 mL of 30 mM Tris-HCl (pH 8.5) overnight to effect intramolecular disulfide cyclization. By adding 5 μL each of 20 mM dithionitrobenzoic acid (DTNB) solution to the peptide solution before the reaction and the peptide solution after the reaction, and confirming that the peptide solution after the reaction is not colored yellow, all the mercapto groups are reacted. Judged that. The cyclized peptide was purified from the reaction solution by reverse phase HPLC.

  Finally, the protected N-terminal cysteine residue was deprotected. The purified peptide was dissolved in 20 mL of water to 1 mM, o-methoxyamine was added to the peptide solution to a concentration of 200 mM, and the reaction was performed at room temperature for 2 days. After completion of the reaction, the desired peptide was obtained by purification with reverse phase HPCL.

2. Preparation method of peptide-immobilized carrier Spherical porous body made of polyvinyl acetate (manufactured by Asahi Glass Co., Ltd., average particle size of 100 μm, exclusion limit molecular weight of about 1 million or more, and the amount of pullulan having a molecular weight of 40,000 that can be filled in 1 mL of resin is 0.00 18 g of 20 mL / mL or more) is charged into 180 mL of dimethyl sulfoxide (Wako Pure Chemical Industries, Ltd.), and 137 mL of epichlorohydrin (Wako Pure Chemical Industries, Ltd.), sodium hydroxide (Wako Pure Chemical Industries, Ltd.) Aqueous solution (21 g / 31.5 mL) and sodium borohydride (Wako Pure Chemical Industries, Ltd.) 88 mg were added and reacted at 30 ° C. for 5 hours to introduce epoxy groups. After the reaction, the spherical porous body into which the epoxy group was introduced was washed with methanol (manufactured by Wako Pure Chemical Industries, Ltd.) and then washed with pure water to obtain an epoxy activated carrier. The obtained carrier was 2 mL of 1.0 M sodium thiosulfate aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd.) and 2 drops of 1% phenolphthalein ethanol solution (manufactured by Wako Pure Chemical Industries, Ltd.) in 1 mL of the activated carrier. Add 0.1 M hydrochloric acid (manufactured by Wako Pure Chemical Industries, Ltd.) until red coloration is not confirmed at 80 ° C., and “activate amount (epoxy group substance amount per unit resin volume) = The amount of the epoxy group introduced by the formula of “hydrochloric acid concentration (volume molar concentration) × hydrochloric acid titration amount (volume amount) / resin amount (volume amount)” was determined and confirmed to be 110 μmol / mL or more.

  Next, 30 mL of the epoxidized carrier was weighed, 45 mL of aqueous ammonia (concentration: 28-30%, Kanto Chemical Co., Inc.) was added, and the mixture was reacted at 40 ° C. for 90 minutes to convert the epoxy group to an amino group. The obtained aminated carrier was quantitatively confirmed to be an amino group from an epoxy group by elemental analysis.

  Next, 5 mL of the amination carrier was weighed and swelled with dimethyl sulfoxide (DMSO, manufactured by Wako Pure Chemical Industries, Ltd.), and then 10 mM Succinimidyl-[(N-maleidopropionamido) -diethyleneglycol] ester (American Thermo Fisher Scientific). 10 mL of DMSO solution) was added and reacted at 30 ° C. for 3 hours. After the reaction, it was washed with DMSO and then washed with pure water to obtain a maleimide carrier.

  The obtained maleimidated carrier was weighed in 1.5 mL, and a 2.4 mM peptide (a peptide consisting of the amino acid sequence of SEQ ID NO: 2, wherein the seventh cysteine residue and the 45th cysteine residue were disulfide to each other. Peptide forming bond) 5 mL of PBS solution was added. Then, the mercapto group in a peptide and the maleimide group in a support | carrier were covalently bound by making it react at 37 degreeC for 17 hours. After the reaction, the peptide-immobilized carrier was obtained by washing with pure water. The amount of peptide immobilization was calculated based on the difference in absorbance after measuring the absorbance (202 nm) of the reaction solution before and after the peptide introduction reaction with an ultraviolet-visible spectrophotometer.

3. Plasma adsorption test CPD-added blood (Citrate Phosphate Dextrose-added blood) collected from healthy individuals was subjected to plasma separation by centrifugation to obtain healthy human plasma. Next, 0.5 mL of the obtained peptide-immobilized carrier was weighed, 6 times its volume of healthy human plasma was added, and the mixture was shaken at 37 ° C. for 17.5. After shaking, the peptide-immobilized carrier is removed by centrifugation, and the plasma protein (IgG, Fbg, albumin (Alb), immunoglobulin M (IgM)) contained in the supernatant plasma is measured to obtain a peptide-immobilized carrier. The adsorption rate to was calculated.

(Comparative Example 1)
10 mL of the epoxidized carrier prepared in Example 1 was weighed, 20 mL of 46 mM tryptophan (manufactured by Wako Pure Chemical Industries, Ltd.) carbonate buffer solution was added, and reacted at 50 ° C. for 16 hours to covalently bind tryptophan to the carrier. It was. After the reaction, a tryptophan-immobilized carrier was obtained by washing with pure water. The amount of tryptophan immobilized was calculated based on the difference in absorbance obtained by measuring the absorbance (280 nm) of the reaction solution before and after the tryptophan introduction reaction with an ultraviolet-visible spectrophotometer. Using this tryptophan-immobilized carrier, a protein adsorption test in healthy human plasma was performed in the same manner as in Example 1.

(Comparative Example 2)
Using the aminated carrier prepared in Example 1, a protein adsorption test in healthy human plasma was performed in the same manner as in Example 1.

The results of Example 1 and Comparative Examples 1 and 2 are shown in Table 1 and FIG.

  As shown in Table 1, Example 1 using a peptide-immobilized carrier showed a high IgG adsorption rate despite the ligand immobilization amount being 1/10 or less of tryptophan. In addition, it was also shown to adsorb very selectively to IgG without adsorbing other plasma protein components as compared to the tryptophan-immobilized carrier.

  The peptide of the present invention and the IgG-type antibody adsorbent using the peptide can be used, for example, in the medical industry and the pharmaceutical industry. In particular, it is possible to adsorb autoantibodies and immune complexes, which are thought to be the etiological agent of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and myasthenia gravis, with high adsorption capacity and high selectivity. Useful in the treatment of autoimmune diseases.

DESCRIPTION OF SYMBOLS 1 ... Body fluid purification device (extracorporeal circulation module) 2 ... Cylindrical 3, 3 '... Filter 4, 4' ... Packing, 5 ... Inlet port (body fluid inlet), 6 ... Cap, 7 ... Outlet port (body fluid guide) Outlet), 8 ... cap, 9 ... IgG type antibody adsorbent.

Claims (7)

A water-insoluble carrier;
A peptide immobilized on the water-insoluble carrier,
The peptide is
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, or
A peptide comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein the seventh cysteine residue and the 45th cysteine residue in the amino acid sequence form a disulfide bond with each other;
The peptide is directly or via a linker by the mercapto group of the 43rd cysteine residue in the amino acid sequence shown in SEQ ID NO: 1 or the mercapto group of the 1st cysteine residue in the amino acid sequence shown in SEQ ID NO: 2. Fixed to the water-insoluble carrier,
IgG type antibody adsorbent. The IgG-type antibody adsorbent according to claim 1 , wherein the linker is oligoethylene glycol or polyethylene glycol. The IgG-type antibody adsorbent according to claim 1 or 2 , wherein the peptide is immobilized in an amount of 0.1 to 100 µmol per mL of the water-insoluble carrier. The IgG-type antibody adsorbent according to any one of claims 1 to 3 , wherein the water-insoluble carrier is a porous body on particles, and the water-insoluble carrier has an exclusion limit molecular weight of 150,000 to 10,000,000. . The IgG type antibody adsorbent according to any one of claims 1 to 4, which is used for a body fluid purification device. A container having an inlet port through which a liquid flows and an outlet port through which the liquid flows out;
An IgG type antibody adsorber comprising the IgG type antibody adsorbent according to any one of claims 1 to 5 filled in the container. A body fluid purification device comprising the IgG type antibody adsorber according to claim 6 ,
A body fluid purification device, wherein the fluid flowing into the IgG antibody adsorber is a body fluid selected from the group consisting of blood, plasma, and serum.

Images